Normal hypothalamic release of gonadotropin releasing hormone (GnRH) is central to regulation of the hypothalamic-pituitary (HP) axis and fertility in mammals including humans. Current therapeutic strategies to control fertility focus on pharmacological control of the HP axis and GnRH. Further; GnRH agonist administration is a key anti-proliferative treatment for some forms of cancer. Our studies of GnRH action define a novel network of cell signaling pathways comprised of calcium and MAPK cascades that target genes essential for the differentiated function of the gonadotrope. Compartmentalized calcium signals are essential for differential activation of MAPKs in gonadotropes. Preliminary studies reveal that specific calcium signals may mediate MAPK activation via calmodulin and the tyrosine kinase, Pyk2. Further, the GnRH receptor and c-raf kinase constitutively reside in specific low density membrane compartments, likely required for assembly of a signaling "module" at the plasma membrane. The specific aims of this proposal are: Aim 1. Define the GnRH signaling pathway in differentiated mouse gonadotropes. This aim seeks to define GnRH signaling mechanisms in fully differentiated gonadotropes, uniquely marked by GFP in transgenic mice. We will examine sexually dimorphic MAPK responses to GnRH and define the gene profile induced by GnRH in gonadotropes. Aim 2: Define the relationship between compartmentalized calcium signals and activation of the ERK and JNK pathways by GnRH. Calcium signaling is required for GnRH-induced ERK activation. Aim 2 examines the role of calmodulin as a calcium sensor and Pyk2 as a calcium-sensitive enzyme that may couple PKC to c-rafkinase activation. Aim 3: Determine the extent and role of compartmentalization of signaling molecules in low-buoyant density membrane rafts in GnRH action. Aim 3 will define the activities present in membrane rafts in gonadotropes, determine the domain requirements for c-raf kinase localization and examine protein-protein interactions that are required for the organization of a putative membrane associated GnRH signaling "module". Aim 4: Define the role of MAPK signaling molecules in GnRH-mediated signaling in vivo. Aim 4 defines the in vivo requirements for ERK l and ERK 2 in the regulation of a GnRH-inducible gene program following ovariectomy in mice. We have obtained three specialized mouse models that provide the unique opportunity to examine the requirement for MAPK signaling within the hvpothalamo-pituitary axis in vivo.